JP5657574B2 - A cell-based assay for the triglyceride synthesis pathway - Google Patents

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[Cross-reference to related applications]
This application claims the benefit of US Provisional Patent Application No. 61 / 149,841, filed Feb. 4, 2009, which is incorporated herein by reference in its entirety and for all purposes. It is.

BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION A method for the simultaneous detection of lipids and phosphatidate intermediates in cells incubated with stable isotope-labeled fatty acids is provided. This method can be used to screen for compounds that modulate triglyceride biosynthesis in a high-throughput format.

Description of Related Art Triglycerides or triacylglycerols are major transport sources and energy storage in eukaryotes. Triglycerides are synthesized from glycerol molecules and three fatty acid molecules. Each fatty acid molecule is attached to the three hydroxyl groups of the glycerol molecule by an ester bond. Triglycerides, like many neutral lipids, contain fatty acid molecules of various chain lengths with typical lengths of 16, 18, or 20 carbons.

式中、ＦＡ−ＣｏＡは、脂肪酸ＣｏＡであり、ＧＰＡＴは、グリセロール−３−リン酸アシルトランスフェラーゼであり、ＡＧＰＡＴは、１−アシルグリセロール−３−リン酸−Ｏ−アシルトランスフェラーゼであり、ＰＡＰは、ホスファチジン酸ホスファターゼであり、ＤＧＡＴは、アシルＣｏＡ：ジアシルグリセロールアシルトランスフェラーゼである。 The two major biosynthetic pathways for triglycerides are the glycerol-3-phosphate pathway that predominates in the liver and adipose tissue, and the monoacylglycerol pathway that predominates in the intestine. The glycerol-3-phosphate pathway that produces more than 90% triglycerides in the liver is exemplified below:

Where FA-CoA is fatty acid CoA, GPAT is glycerol-3-phosphate acyltransferase, AGPAT is 1-acylglycerol-3-phosphate-O-acyltransferase, and PAP is Phosphatidate phosphatase, DGAT is an acyl CoA: diacylglycerol acyltransferase.

The final step of the glycerol-3-phosphate biosynthetic pathway can be catalyzed by DGAT1 or DGAT2 (Cases et al. 1998, Proc Natl Acad Sci USA 95: 13018; Cases et al. 2001 J Biol Chem 276: 38870). Both DGAT1 and DGAT2 convert diglycerides to triglycerides, but they are not similar in nucleotide or amino acid sequence. Knockout mice without DGAT1 (Dgatl ^{− / −} ) have been reported to show no apparent changes in triglyceride metabolism in the liver (Smith et al. 2000, Nat Genet 25:87). In addition, knockout mice without DGAT2 (Dgat2 ^{− / −} ) show a dramatic decrease in triglyceride content in the liver (Stone et al. 2004, J Biol Chem 279: 11767). In addition, studies have shown that DGAT2 inhibition by antisense oligonucleotides reduces liver triglyceride content in rodents (Chol et al. 2007, J Biol Chem 282: 22678; Liu et al. 2008, Biochim Biophy Acta 1781: 97) have been shown to reverse diet-induced fatty liver and insulin resistance in rats (Chol et al. 2007, J Biol Chem 282: 22678). These studies suggest that DGAT1 and DGAT2 function differently in triglyceride biosynthesis. Thus, specific targeting of either DGAT1 or DGAT2 can provide benefits in the regulation of triglycerides with limited toxicity.

  Disturbances or imbalances in triglyceride metabolism are associated with the pathogenesis and increased risk of obesity, metabolic syndrome, type II diabetes, nonalcoholic fatty liver disease, and coronary heart disease (Lewis et al. 2002, Endocrine Reviews 23 : 201; Malloy and Kane 2001, Adv Intern Med 47: 111). Thus, compounds that modulate enzyme activity within the triglyceride biosynthetic pathway, including DGAT1 and DGAT2, may be useful as potential therapeutic targets for metabolic diseases.

Radioactive substrates and thin layer chromatography are widely used to observe triglyceride synthesis (Stone et al. 2004, J Biol Chem 279: 11767; Zhu et al. 2002, Atherosclerosis 164: 221). Stone et al. Label triglycerides with ³ H-glycerol in primary cultured hepatocytes and detect radioisotope-labeled triglycerides using thin layer chromatography and radio image analysis (Stone et al. 2004, J Biol Chem 279: 11767). Similarly, Zhu et al. Labeled triglycerides with ³ H-oleic acid in human hepatocellular carcinoma cells and detected the labeled triglycerides using thin layer chromatography (Zhu et al. 2002, Atherosclerosis 164: 221).

Recently, Magkos et al. Have detected neutral lipids using mass spectrometry technology (Magkos et al. 2007, J Lipid Research 48: 1204). Magkos et al, ^1,1,2,3,3-2 H- glycerol and ^2,2-2 H- palmitic acid administered in in vivo, by chloroform / methanol and ultracentrifugation, very low-density Lipoprotein triglycerides are extracted from plasma. Magkos et al. Used a gas chromatography-mass spectrometry system to detect labeled glycerol and methylated palmitic acid released from triglycerides (Magkos et al. 2007, J Lipid Research 48: 1204). Magkos et al. Have not detected unchanged triglycerides, ie, specific triglyceride molecules, nor different intermediates in the triglyceride pathway.

  To the best of Applicants' knowledge, existing assays do not observe or detect intermediates including lysophosphatidic acid, phosphatidic acid, and diacylglycerol produced during triglyceride biosynthesis. Existing assays also require additional extraction procedures and labor intensive detection techniques. Furthermore, these methods have limited throughput and are difficult to adapt to high-throughput formats for screening large numbers of compounds. Thus, there remains a need to establish methods to analyze triglyceride biosynthesis and identify compounds that modulate the triglyceride pathway in a high-throughput format.

〔Overview〕
The purpose of this application is to provide a method for identifying compounds that inhibit the activity of acyl CoA: diacylglycerol acyltransferase (DGAT). This method comprises the steps of contacting a stable isotope-labeled fatty acid with a cell in the presence or absence of the compound, extracting the cell with isopropyl alcohol, and stable isotope-labeled triglyceride in the presence of the compound. And measuring the level of stable isotope-labeled triglyceride in the absence of the compound, wherein the change in the level of stable isotope-labeled triglyceride causes the compound to inhibit DGAT activity. Indicates.

  Another object of the present application is to provide a method for measuring the activity of enzymes involved in triglyceride synthesis. This method detects the presence of a stable isotope-labeled fatty acid molecule in contact with a cell, the step of extracting the cell with isopropyl alcohol, and the presence of a stable isotope-labeled lysophosphatidic acid, phosphatidic acid, diacylglycerol, or triglyceride. And a process.

According to the present application, the stable isotope labeled fatty acid may be ¹³ C ₁₈ -oleic acid. Also according to the present application, stable isotope labeled triglycerides can be detected by a liquid chromatography-mass spectrophotometer system.

  Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is understood, however, that the drawings are designed for illustrative purposes only and are not designed as a definition of the limits of the invention to be referred to the claims. The drawings are not necessarily drawn to scale, and unless otherwise specified, the drawings are only intended to conceptually describe the structures and procedures described herein, It should be further understood.

[Detailed Description of Presently Preferred Embodiment]
The present application provides a method to overcome the limitations of existing assays for analyzing triglyceride synthesis. The methods described herein use stable isotope-labeled fatty acids in the reaction mixture, such that the reaction mixture contains cells, growth media, and stable isotope-labeled fatty acids. The methods described herein can also be used to screen for compounds that modulate enzymes involved in triglyceride synthesis. For screening purposes, the reaction mixture may further comprise a modulator of enzymes involved in triglycerides, such as DGAT in the glycerol-3-phosphate pathway.

  Any prokaryotic and eukaryotic cell can be used in the methods described herein. Adipocytes, hepatocytoma cells, cancer cells, or leukemia cells are preferred, such as in 3T3-L1, HUH7, FU5AH, THP-1, HepG2, C3HT101 / 2, McA-RH7777, MCF7, A549, and RAW264.7 is there. These cells are obtained through commercial sources or can be isolated by commonly known methods. For example, a method for isolating hepatocytes can be found in Berry and Friend, 1969, J Cell Biol 43: 506, and a method for isolating adipocytes can be found in Green et al. 1990, J Biol Chem 265: 5206. Can be seen. Leukocytes can be isolated from biological samples taken from an individual, such as any body fluid or tissue sample. Any bodily fluid includes, but is not limited to, serum, plasma, lymph, cyst fluid, urine, stool, cerebrospinal fluid (csf), and ascites fluid. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, and biopsies of specific organ tissues such as muscle or nerve tissue and hair. Analysis of the activity of the glycerol-3-phosphate pathway in biological samples can be useful as a biomarker to observe patient response to treatment with drugs for metabolic diseases.

  Cells can be maintained in normal growth media such as Dulbecco's Modified Eagle's Medium, Minimum Essential Medium, RPMI, and the like. Some cells can be induced or differentiated before being analyzed. One skilled in the art will recognize that appropriate media and conditions are required to maintain and / or induce specific cell lines. For example, 3T3-L1 preadipocytes can be maintained in Dulbecco's modified Eagle medium and induced to become mature adipocytes by induction medium.

Any fatty acid molecule labeled with a stable isotope can be used in the methods described herein. As used herein, the term “stable isotope” generally refers to a non-radioactive isotope molecule comprising ¹³ C or ² H. Stable isotope-labeled fatty acids can be synthesized indoors or obtained commercially. Suitable fatty acid molecules, such as monounsaturated _{C 16} and _{C 18,} comprising a chain length ranging from C 10 _{-C 20.} Fatty acid molecules may be uniformly or partially labeled with ¹³ C or ² H, for example, uniformly labeled ¹³ C-14: 0, ¹³ C-16: 1, ¹³ C-16: 0. , ¹³ C-18: 2, ¹³ C-18: 1, ¹³ C-18: 0, ² H ₂₉ -16: 1, ² H ₃₁ -16: 0, ² H ₃₃ -18: 1, ² H _35- 18: 0, or partially labeled ² H with a mass shift of at least 4 amu (6.64 × 10 ⁻²⁷ kg). In one embodiment, the labeling molecule is ¹³ C ₁₈ -oleic acid. Stable isotope-labeled fatty acids can be incorporated into each step of the pathway to yield new synthetic intermediates, or products labeled with stable isotopes.

  A variety of other reagents can also be included in the reaction mixture. These include reagents such as salts, buffers, neutral proteins (eg, albumin), detergents, and the like that can be used to promote enzyme activity. Such reagents can also reduce non-specificity or background interactions of reaction components. Other reagents that improve the efficiency of the assay, such as inhibitors, antibacterials, etc., can also be used.

Prior to initiating the method of analyzing triglyceride biosynthesis, endogenous fatty acids are depleted by culturing the cells in charcoal-treated serum or serum-free medium for an appropriate period of time. Next, a stable isotope labeled fatty acid, such as ¹³ C ₁₈ -oleic acid, is added to the reaction. The cells are then washed with phosphate buffered saline and lipid molecules are extracted with isopropyl alcohol. Other solvent systems including hexane, chloroform, heptane, acetone, dimethyl sulfur oxide, and butanol can also be used to extract lipid molecules.

  The newly synthesized labeled lipid intermediate or product can be detected by any method suitable for detecting the presence of an isotope or measuring the molecular mass of an isotope molecule. For example, thin layer chromatography and mass spectrometry can be used. In one embodiment, a liquid chromatography-mass spectrometry (LC-MS) system is used to detect and quantify newly synthesized lipid molecules comprising stable isotope labeled acyl chains. Furthermore, a plurality of labeled lipid molecules can be detected simultaneously. In one embodiment, the mass spectrometry system is modified for simultaneous analysis of multiple neutral lipids, including monoacylglycerol, diacylglycerol, and triacylglycerol and a phosphatidic acid intermediate in the glycerol-3-phosphate pathway. Is done. Modifications include work by electrospray ionization in positive and negative switching without pre-column derivatization in the mass spectrometry system. Simultaneous detection of a plurality of lipids can also be performed in a single analysis operation.

  Once the labeled lipid molecules are detected, their appropriate level can be measured by a normalized concentration method. As an example, the normalized concentration is measured by an internal standard of 1,3-dipalmitoyl-2-stearoyl-glycerol-d5 (PSP-d5). The relative response of triolein or newly synthesized lipid molecules to that of the internal standard in the sample is obtained and used for quantification. It can be seen here that an approximately 1 μM solution of PSP-d5 in isopropyl alcohol can be used directly to extract lipid molecules. In addition, an ammonium adduct ion of PSP-d5 at m / z 858.0 can be used for ion extraction and incorporation. One skilled in the art can measure a particular level of the detection molecule by other commonly used methods.

  To assess whether a compound can modulate triglyceride synthesis, the compound is added to a reaction mixture containing stable isotope labeled fatty acids, growth media and cells. The reaction is incubated for a suitable time, for example from about 10 minutes to about 24 hours. The reaction is extracted for lipid molecules with isopropyl alcohol and analyzed using an LC-MS system. If the level of labeled triglyceride in the reaction with the compound is significantly different from that in the reaction without the compound, the compound appears to regulate the enzymes involved in triglyceride synthesis. As used herein, the term “significantly different” means that one skilled in the art will think that the difference modulates triglyceride synthesis. The difference in the level of labeled triglyceride may be at least 10%, preferably at least 25%.

  The compounds encompass many chemical classes, but are typically organic compounds. The compound also contains functional chemical groups necessary for structural interaction with the polypeptide, typically containing at least an amine group, a carbonyl group, a hydroxyl group, or a carboxyl group, preferably those. At least two of the chemical functional groups, and more preferably at least three of the chemical functional groups. The compound can include a cyclic carbon or heterocyclic structure and / or an aromatic or polycyclic aromatic structure substituted with one or more of the aforementioned functional groups. The compounds may be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structure analogs of the foregoing, or combinations thereof. Where the compound is a nucleic acid, the compound is typically a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.

  The compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Many means are available for random and direct synthesis of a wide range of organic compounds and biomolecules including, for example, expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Compounds are biological libraries; combinatorial library methods known in the art including spatially addressable parallel solid phase or solution phase libraries: deconvolution integration Can be obtained using any of a number of approaches in synthetic library methods that require the use of: "one bead one compound" library method; and synthetic library methods using affinity chromatography selection (Lam 1997, Anticancer Drug Des. 12: 145). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily generated. Furthermore, natural and synthetically generated libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means.

  In addition, known drugs have undergone directed or random chemical modifications, such as acylation, akylation, esterification, amidification, etc., and their structural similarity The body can be generated. The compounds may be selected randomly or may be based on existing compounds that bind to and / or modulate DGAT activity. Thus, the candidate drug source is a library of molecules based on known DGAT modulators, and the structure of the compound contains more or less chemical moieties or different chemical moieties, It varies at one or more positions in the molecule. The structural changes made to the molecules in creating the analog activator / suppressor library can be directed, random, or both directed and random substitution and / or addition. Can be a combination of Those skilled in the art of preparing combinatorial libraries can readily prepare such libraries based on existing DGAT modulators. As used herein, the terms “modulator”, “modulate” and the like refer to a compound that increases or decreases enzyme activity. If the enzyme activity decreases in the presence of the compound, the compound is called an inhibitor. If the enzyme activity increases in the presence of the compound, the compound is called an activator.

化合物２、すなわちＤＧＡＴ２抑制物質は、ジメチル１−［シクロヘキシル（３，４−ジクロロフェニル）メチル］−２−チオキソ−２，３−ジヒドロ−１Ｈ−イミダゾール−４，５−ジカルボキシレートである。化合物２は、４５７．３８の分子量を有し、以下の構造を有する。

By way of example, the effect of DGAT modulator compounds 1 and 2 on triglyceride synthesis is evaluated using the methods described herein. Compounds 1 and 2 are disclosed in WO2008148868 and WO2004069809, which are incorporated by reference in their entirety. Compound 1, a DGAT1 inhibitor, is N- [2,6-dichloro-4- (pyrrolidin-1-ylmethyl) phenyl] -4- (4-{[(4-methoxyphenyl) acetyl] amino} phenyl) piperazine -1-carboxamide. Compound 1 has a molecular weight of 596.55 and has the following structure:

Compound 2, the DGAT2 inhibitor, is dimethyl 1- [cyclohexyl (3,4-dichlorophenyl) methyl] -2-thioxo-2,3-dihydro-1H-imidazole-4,5-dicarboxylate. Compound 2 has a molecular weight of 457.38 and has the following structure:

  If the LC-MS system is used for detection of labeled triglyceride molecules, a suitable solution is needed to extract lipid molecules from the reaction mixture. Some organic solvents such as hexane, chloroform / methanol, DMSO, etc. may not be compatible with a reverse phase LC-MS detection system. Thus, when using hexane, chloroform / methanol, DMSO, etc. for extraction, remove such organic solvents and reconstitute the extraction reaction mixture with a compatible solvent system prior to detection by the LC-MS system. This stage is necessary.

  Since it has been found herein that the use of isopropyl alcohol is compatible with LC-MS systems, isopropyl alcohol can be used to extract and deliver lipid molecules without additional steps. One-step extraction increases efficiency and enables a high throughput format desirable for screening and identifying compounds that modulate triglyceride synthesis.

  The term “high throughput” refers to an assay design that allows easy screening of multiple samples at the same time and the capability of robotic manipulation. Another desired feature of high-throughput assays is an assay design that is optimized to achieve the desired analysis by reducing reagent usage or minimizing the number of manipulations. is there. Examples of assay formats include 96-well or 384-well plates, levitating droplets, and “lab on a chip” microchannel chips used for liquid handling experiments. It is well known in the art that more and more sampling can be performed using the design of the present invention as plastic molds and liquid handling devices are miniaturized or as improved assay devices are designed. It is.

  In one embodiment, the cells are cultured and analyzed in a microtiter plate comprising a plurality of wells, such as a 96 or 386 well plate. The plates are loaded into a detection system such as LC-MS that reads the reaction in each well of each plate and generates numerical data. The methods described herein are also useful for assessing the cellular activity of enzymes involved in triglyceride synthesis, including DGAT, GPAT, AGPAT, and PAP. This method may also be used to screen for compounds that modulate enzyme activity in triglyceride biosynthesis in a high-throughput format. Furthermore, the methods of the present application can be used as a diagnostic assay to measure the metabolic activity of triglyceride synthesis in isolated cells from human subjects.

Example 1 Cell Line Preparation 3T3-L1 mouse preadipocytes were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were seeded in 96 well plates. Two days after confluency, the medium was changed to induction medium (Adipolysis kit, Cayman Chemical Company) containing IBMX, insulin, and dexamethasone. Three days after induction, the medium was changed to insulin medium (Adipolysis kit, Cayman Chemical Company). Five days after induction, the medium was replaced with fresh insulin medium for 2 days. The human hepatoma cell line HUH7 was maintained in DMEM containing 10% FBS, 4.5 g / L D-glucose. The rat hepatoma cell line FU5AH was maintained in Eagle's minimum essential medium (MEM) containing 5% FBS. Hepatoma cells were seeded in 96-well plates and used at 80% confluence. Human breast cancer cell line MCF7 cells were maintained in DMEM with 10% FBS, 4.5 g / L D-glucose. The human monocytic leukemia cell line THP-1 was maintained in RPMI medium containing 10% FBS and used at 5 × 10 ⁵ cells / mL.

Example 2 Stable Isotope Labeling of Triglyceride Synthesis Pathway HUH7 cells or differentiated 3T3-L1 cells are treated with 10% charcoal-stripped FBS (Gibco) in 5% CO ₂ overnight at 37 ° C. Incubated with DMEM containing FU5AH cells were incubated with MEM containing 5% charcoal stripped FBS overnight at 37 ° C. in 5% CO ₂ . About 100 μL of 300 μM ¹³ C ₁₈ -oleic acid (SIGMA) pre-complexed with 0.5% fatty acid-free BSA (SIGMA) is 0, 30, 60 at 37 ° C. in 5% CO _2. , 120 and 180 minutes added to the cell suspension. The cells are then washed once with phosphate buffered saline and extracted with 100 μL isopropyl alcohol. After incubating for 10 minutes at room temperature, the extract was transferred to a glass vial and detected for labeled lipid molecules using an LC-MS system.

LC-MS detection was performed using an Agilent 1100 Liquid Chromatographic system (Agilent Technologies, Palo Alto, CA) coupled with a Micromass triple-quadrupole Quattro Micro mass spectrophotometer (Waters, Milford, Mass.) Through a Z-spray electrospray ion source (ESI). ) Was executed. Metabolite separation was performed on an Eclipse XDB-C ₈ column (2.1 × 50 mm, particle size = 3.5 μm). Mobile phase A, 50 mM ammonium formate in isopropanol-water (3: 7), and mobile phase B, 10 mM ammonium formate in isopropanol-water (9: 1) were used for gradient elution as follows. : 20-100% mobile phase B for 1 minute, 100% mobile phase B retention for 4 minutes, return to 20% mobile phase B for 0.1 minute, after 4 post run times .5 minutes. The flow rate was 0.5 mL / min. The resulting LC eluent was introduced into the mass spectrometer at 0.25 mL / min in a 1: 2 split. The mass spectrometer was operated in positive and negative ion switching mode. Nitrogen was used as the nebulizing gas, the desolvation gas, and the cone curtain gas. The source parameters of the MS system are: capillary voltage, 3.1 kV; cone voltage, 20 V (ESI ⁺ ) and 15 V (ESI ⁻ ); extractor, 2 V; RF lens, 0.1 V; source temperature ( source temperature), 120 ° C; desolvation temperature, 300 ° C; LM1, HM1, LM2 or HM2 resolution, 15; ion energy, 1.0; entrance & exit, 15. MassLynx software version 4.1 was used for system control and data processing.

In FU5AH cells, ¹³ C ₁₈ to glycerol-3-phosphate pathway - oleic acid ^uptake, 13 _{C 18} - was determined by the presence of triolein (Figure 1). The level of ¹³ C ₁₈ -triolein increased with incubation time by comparing to the area under the curve at 30 min, 60 min, 120 min, and 180 min (FIG. 1). Further, intermediates of oleoyl lysophosphatidic acid, 2-mono-oleoyl glycerol, 1,2-dioleoyl-sn-glycero-3-phosphatidic acid, 1,2-dioleoyl-sn-glycerol using positive / negative ion switching A new LC-MS method was developed here for the simultaneous detection of trioleoyl-sn-glycerol end product (FIG. 2). This indicates that stable isotope labeling combined with detection of the LC-MS system can be used for simultaneous detection of various stages of triglyceride biosynthesis in cells.

Example 3 Evaluation of Compounds that Modulate DGAT Activity To evaluate compounds that modulate DGAT1 or DGAT2 activity, HUH7 or 3T3-L1 cells were treated with 100 μL containing 0.05% DMSO for about 10 minutes at 37 ° C. In serum-free medium with about 0, 0.03, 0.1, 0.3, 1, 3, or 10 μM of compound 1 or 2. Controls are cells incubated with 100 μL of serum-free medium containing 0.05% DMSO for about 10 minutes at 37 ° C. in the absence of any compound. About 100 μL of 600 μM ¹³ C ₁₈ -oleic acid previously synthesized with 1.0% fatty acid free BSA was added to the cell suspension. After incubation for about 90 minutes at 37 ° C., cells were extracted and detected as described in Example 2.

The influence of compounds 1 and 2 on triglyceride biosynthesis is shown in FIGS. Both HUH7 and the differentiated 3T3-L1 cell line had reduced levels of newly synthesized ¹³ C ₁₈ -triolein in the presence of Compound 1. In HUH7 cell lines, ¹³ C ₁₈ in cells incubated with Compound 1 10 [mu] M - triolein level is about 30% of the control, ¹³ in cells incubated with compound 2 in 10 [mu] M C ₁₈ - Trio The level of rain was about 75% of the control (Figure 3). In addition, a transient increase in ¹³ C ₁₈ -oleoyl-diglyceride was detected in HUH7 cells incubated with Compound 1 (FIG. 5). These results suggest that HUH7 cells have both functional DGAT1 and DGAT2 enzymes that convert ¹³ C ₁₈ -diolein to ¹³ C ₁₈ -triolein.

In the differentiated 3T3-L1 cell line, the level of ¹³ C ₁₈ -triolein in cells incubated with about 10 μM Compound 1 was about 33% of the control (FIG. 4). This result suggests that in differentiated 3T3-L1 cells, most ¹³ C ₁₈ -triolein is synthesized by DGAT1.

Next, the effect of Compound 2 in MCF7 cells was evaluated. MCF7 cells were seeded in DMEM containing 10% FBS, 4.5 g / L D-glucose in 96-well culture plates for 24 hours. Confluent or nearly confluent cells were incubated for 1 hour with about 150 μL of serum-free DMEM containing 0.15% fatty acid-free BSA. Next, about 3 μL of Compound 2 in DMEM containing 3% DMSO was added to a final concentration of about 0, 0.1, 0.3, 1, 3, 10 μM. After incubation at 37 ° C for 15 minutes, the plate was drained. The cells are then placed in DMEM containing 3% DMSO for about 3 hours with 0.15% fatty acid free BSA and 150 μL of 100 μM ¹³ C ₁₈ -oleic acid and about 3 μL of Compound 2 pre-synthesized. And incubated at 37 ° C. The cells were extracted with approximately 100 μL of isopropyl alcohol followed by LC-MS detection as described above. As shown in FIG. 6, the level of ¹³ C ₁₈ -triolein in cells incubated with compound 2 was approximately 80% of the control. This result suggests that the majority of ¹³ C ₁₈ -triolein is synthesized by DGAT2 in the MCF7 cells used here.

In the glycerol-3-phosphate pathway, additional experiments were performed to detect the presence of labeled intermediate as the production of ¹³ C ₁₈ -triolein with three labeled fatty acid chains with other enzymes. FU5AH cells were incubated with ¹³ C ₁₈ -oleic acid for 60 minutes and analyzed using simultaneous detection as described in Example 2. As shown in FIG. 7, trioleins containing one, two, and three ¹³ C ₁₈ -oleoyl side chains are labeled with molecular masses of m / z 921.0, 939.0, and 957.0, respectively. Appears as a molecule.

  The results of Example 3 indicate that differentiated adipocytes and HUH7 cells can be used to screen for compounds that modulate DGAT1 activity. On the other hand, MCF7 cells can be used to screen for compounds that modulate DGAT2 activity. In addition, MCF7, adipocytes, and hepatoma cells have been used to screen compounds that modulate other enzymes, including GPAT, AGPAT, or PAP involved in triglyceride biosynthesis, such as the glycerol-3-phosphate pathway. Good.

Example 4 Detection of triglyceride biosynthesis in leukocytes To determine whether triglyceride biosynthesis is present in leukocytes, THP-1 cells were incubated with serum-free RPMI and incubated at 37 ° C for 2 hours. Approximately 200,000 cells were centrifuged at 500 xg for 5 minutes. In RPMI, 0.5% fatty acid-free BSA (SIGMA) and approximately 300 μL of 300 μM ¹³ C ₁₈ -oleic acid (SIGMA) previously synthesized were added to the cell pellet. The cells were incubated with the labeled molecule for 2 hours at 37 ° C. and extracted with isopropyl alcohol as described above.

As shown in FIG. 8, newly synthesized ¹³ C ₁₈ -triolein was detected in THP-1 cells cultured with labeled oleic acid but not in control cells cultured without labeled oleic acid. There wasn't. When THP-1 cells were incubated with ¹³ C ₁₈ -oleic acid for about 120 minutes, triolein with one or two ¹³ C ₁₈ -oleoyl side chains was detected as a labeled molecule, and the molecular mass was M / z 921.0 and 939.0, respectively.

  Detection of partially labeled triolein indicates that the method of the present application can be used to detect cellular activity of AGPAT, PAP, or DGAT. Furthermore, the methods described herein may be used to detect newly synthesized triglycerides in isolated cells and thus can be used as a diagnostic assay to measure the metabolic activity of triglyceride biosynthesis.

  All references cited in this section are incorporated by reference in their entirety.

  Thus, while applied to the preferred embodiment, the fundamental novel features of the present invention have been illustrated, described, and pointed out, although various forms and details of the apparatus illustrated and their operation may be used. It will be understood that omissions, substitutions, and changes may be made by those skilled in the art without departing from the spirit of the invention. For example, all combinations of elements and / or method steps that perform substantially the same function in substantially the same way to obtain the same result are expressly intended to be within the scope of the invention. . Further, the structures and / or elements and / or method steps illustrated and / or described in connection with any disclosure form or embodiment of the present invention may be considered as a general matter of design choice as any other disclosure form. It should be appreciated that the description, or suggestion or embodiment may be incorporated into the embodiment. Accordingly, it is intended to be limited only as indicated by the scope of the claims.

Embodiment
(1) In a method for identifying a compound that suppresses the activity of acyl CoA: diacylglycerol acyltransferase (DGAT),
a. Contacting the cell with a stable isotope-labeled fatty acid in the presence and absence of the compound;
b. Extracting the cells with isopropyl alcohol;
c. Measuring the level of stable isotope-labeled triglycerides in the presence of the compound;
d. Measuring the level of stable isotope-labeled triglycerides in the absence of said compound;
Including
A change in the level of the stable isotope labeled triglyceride indicates that the compound inhibits DGAT activity.
(2) In the method according to embodiment 1,
The method wherein the stable isotope is ¹³ C.
(3) In the method according to embodiment 1,
The method wherein the stable isotope-labeled fatty acid is ¹³ C ₁₈ -oleic acid.
(4) In the method according to embodiment 1,
The stable isotope labeled triglyceride is detected by a liquid chromatography-mass spectrophotometer system.
(5) In a method for measuring the activity of an enzyme involved in triglyceride synthesis,
a. Contacting the cell with a stable isotope-labeled fatty acid;
b. Extracting the cells with isopropyl alcohol;
c. Detecting the presence of stable isotope-labeled lysophosphatidic acid, phosphatidic acid, or diacylglycerol or triglycerides;
Including a method.

(6) In the method according to embodiment 5,
The method wherein the enzyme is acyl CoA: diacylglycerol acyltransferase (DGAT).
(7) In the method according to embodiment 5,
The method wherein the enzyme is glycerol-3-phosphate acyltransferase (GPAT).
(8) In the method according to embodiment 5,
The method wherein the enzyme is 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT).
(9) In the method according to embodiment 5,
The method wherein the enzyme is phosphatidate phosphatase (PAP).
(10) In the method according to embodiment 5,
The method wherein the stable isotope is ¹³ C.

(11) In the method according to embodiment 5,
The method wherein the stable isotope-labeled fatty acid is ¹³ C ₁₈ -oleic acid.

3 is an extracted ion chromatogram of ¹³ C-labeled triolein having ¹³ oleoyl- ¹³ C ₁₈ side chains, which is 0 min (A), 30 min (B), 60 min (C), 120 min (D). And the overall uptake of free ¹³ C ₁₈ -oleic acid into triglycerides in FU5AH cells at time points 180 and (E). Simultaneous observation of intermediate and final product of triolein synthesis using LC-MS positive and negative ion switching: (A) oleoyl lysophosphatidic acid, (B) monooleoyl glycerol, (C) dioleoylphosphatidic acid ( dioleoyl phosphatidyate), (D) dioleoylglycerol, and (E) trioleoylglycerol. Three oleoyl of HUH7 hepatocytes ^- for new synthetic triolein having a 13 _{C 18} chain, evaluation of the effect of DGAT1 and DGAT2 inhibitors. Oleoyl three 3T3-L1 differentiated adipocytes ^- for new synthetic triolein having a 13 _{C 18} chain, evaluation of DGAT1 inhibitor. HUH7 during DGAT1 inhibition of liver cells, two oleoyl ^- transient increase in diglycerides with 13 _{C 18} chain. Three oleoyl of MCF7 cells ^- for new synthetic triolein having a 13 _{C 18} chain, evaluation of DGAT2 inhibitors. Oleic 60 minutes ^- after incubation at 13 _{C 18} acid (oleic- ¹³ C ₁₈ acid), one of FU5AH cells, two, and three oleoyl ^- 13 triolein simultaneous detection of having _{C 18} side chains : (A) endogenous triolein detected at m / z 903.0, (B) ¹³ C ₁₈ -triolein detected at m / z 921.0, (C) ¹³ detected at m / z 939.0 C ₃₆ -triolein and (D) ¹³ C ₅₄ -triolein detected at m / z 957.0. ¹³ C ₁₈ - together with oleic acid or ¹³ C ₁₈ - oleic acid without at the incubated THP-1 in monocytes 120 minutes, one, two, and three oleoyl ^- trio with 13 _{C 18} side chains rain simultaneous detection: ^(a) 13 _{C 18} - ^triolein, 13 _{C 18} - in the absence of oleic acid was not detected in ^{m / z921.0; (B) 13} C 18 - ^{triolein, 13} C ₁₈ - was observed in the presence of oleic acid; _^(C) 13 ^C ₃₆ - ^triolein, 13 _{C 18} - in the absence of oleic acid was not detected in m / z939.0; ^{(D) 13} C ₃₆ -triolein was observed in the presence of ¹³ C ₁₈ -oleic acid; (E) ¹³ C ₅₄ -triolein was observed in the absence of ¹³ C ₁₈ -oleic acid. Not detected at m / z 957.0; (F) ¹³ C ₅₄ -triolein was observed in the presence of ¹³ C ₁₈ -oleic acid.

Claims (11)

In a method for identifying a compound that inhibits the in vivo activity of acyl CoA: diacylglycerol acyltransferase (DGAT) in a cell ,
a. Contacting the cell with a stable isotope-labeled fatty acid in the presence and absence of the compound;
b. Extracting the cells with isopropyl alcohol;
c. Measuring the level of stable isotope-labeled diacylglycerol and triglyceride in the presence of said compound;
d. Measuring the level of stable isotope-labeled diacylglycerol and triglyceride in the absence of said compound;
e. Comparing the level of stable isotope-labeled diacylglycerol and triglyceride in the presence of said compound with the level of said stable isotope-labeled diacylglycerol and triglyceride in the absence of said compound;
Including
A method wherein an increase in the level of stable isotope-labeled diacylglycerol and a decrease in the level of stable isotope-labeled triglyceride in the presence of said compound indicates that said compound inhibits DGAT activity. The method of claim 1, wherein
The method wherein the stable isotope is ¹³ C. The method of claim 1, wherein
The method wherein the stable isotope-labeled fatty acid is ¹³ C ₁₈ -oleic acid. The method of claim 1, wherein
The stable isotope labeled triglyceride is detected by a liquid chromatography-mass spectrophotometer system. In a method for measuring the in vivo activity of an enzyme involved in triglyceride synthesis in a cell ,
a. Contacting the cell with a stable isotope-labeled fatty acid;
b. Extracting the cells with isopropyl alcohol;
c. Detecting the presence of stable isotope-labeled lysophosphatidic acid, phosphatidic acid, or diacylglycerol or triglycerides in the extract ;
Only including,
A method wherein the presence of the stable identification labeled lysophosphatidic acid, phosphatidic acid, diacylglycerol or triglyceride indicates the activity of the enzyme . The method of claim 5, wherein
The method detects the presence of stable isotope labeled triglycerides,
The method wherein the enzyme is acyl CoA: diacylglycerol acyltransferase (DGAT). The method of claim 5, wherein
The method detects the presence of stable isotope labeled lysophosphatidic acid,
The method wherein the enzyme is glycerol-3-phosphate acyltransferase (GPAT). The method of claim 5, wherein
The method detects the presence of stable isotope labeled phosphatidic acid,
The method wherein the enzyme is 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT). The method of claim 5, wherein
The method detects the presence of stable isotope labeled diacylglycerol,
The method wherein the enzyme is phosphatidate phosphatase (PAP). The method of claim 5, wherein
The method wherein the stable isotope is ¹³ C. The method of claim 5, wherein
The method wherein the stable isotope-labeled fatty acid is ¹³ C ₁₈ -oleic acid.

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